Transcriptional regulation of gene expression is a complex task, critical for growth and survival, whether as part of the developmental process from the fertilized egg, or when adapting to changing environmental conditions. The transcription initiation step is arguably the most regulated step in gene transcription, as fine tuning both its rate and synchrony can serve as a key control point to produce organism-wide changes in gene expression profiles in response to developmental and environmental cues. Not surprisingly, the complexity of gene regulatory circuitries is paralleled by the size and complexity of the molecular players involved in transcription initiation. Over the past 30 years, biochemistry, molecular genetics, and in vivo studies have uncovered most, if not all, of the central components of the transcriptional apparatus. However, a mechanistic understanding of gene expression in humans poses a formidable challenge and lags dramatically behind. A major obstacle is that the transcriptional machinery comprises more than 100 individual polypeptides that operate as a huge and dynamic assemblage made up of functionally distinct multi-subunit complexes, many of them only accessible from endogenous sources. We are using single particle EM reconstruction to characterize the architecture, dynamics and interactions of large human complexes essential for gene regulation. 3D Cryo-EM reconstruction is a technique ideally suited to this task, as it requires limited amounts of material, is optimal to study very large assemblies, and is has the potential to detect and characterize conformational flexibility. The latter is a property that may prove critical to be able to describe the functional plasticity required in transcriptional complexes like TFIID, an essential hub in this process that needs to bind to different DNA core promoters and integrate the input from a large variety of transcriptional activators and cofactors. The significance to human health of a fundamental understanding of how gene transcription is switched on and off cannot be overstated. Such significance is highlighted by the fact that development of various cancers is accompanied by alterations in gene expression leading to various aspects of the disease, and by the discovery of induced pluripotent stem cells, where the expression of 3-4 global transcriptional activators is sufficient to introduce gene expression changes needed to transition into a pluripotent state. By understanding the protein structures necessary for gene activation, we will guide future research into the development of novel treatments that target the control of gene expression mediating cell growth, neoplasia, metastasis, and angiogenesis in humans or those aimed at facilitating the transition of induced pluripotent stem cells research into the clinic.